Ruminant feed composition containing an amidine



animal.

Patented Mar. 3, 1953 RUMINANT FEED COMPOSITION CON- TAINING AN AMIDINE Kenneth E. Walker, Wilmington, Del., assignor to E. I. du Pont de Nemours and Company, Wilmington, Del., a corporation of Delaware No Drawing. Application November 3, 1951, Serial No. 254,787

10 Claims. (Cl. 99-2) This invention relates to a new source of nitrogen to supplement the protein feed of Ruminantia, a division of ungulate mammals, with a quadripartite stomach and more especially is directed to providing certain amidines as a source of usable nitrogen to supplement the protein feeds for such animals.

Burroughs et al., of the Ohio Agricultural Experimental Station, have called attention to the importance of nutrients neededby rumen microorganisms in the digestion of roughages and particularly the cellulose fraction of their normal feed. (Jour. Animal Science, 9, 523-530, 1950.) From the fact that the feeding of cattle and other ruminant animals often includes the feeding of grains and protein-rich feeds in combination with roughage, Burroughs has postulated that these grains and protein-rich feeds, when fed with roughage, might supply nutritional factors needed by rumen microorganisms in the digestion of the roughage fraction of the ration (Feedstuffs, p. 24, 12-9-50). These investigators present considerable evidence supporting the proposition that grains and other protein-rich feeds contribute to the nutrition of rumen microorganisms in their digestion of cellulose.

Further work has established that urea is a source of usable nitrogen which may replace a portion, at least, of the protein feed for such animals. By usable nitrogen is meant the nitrogen of a compound that is a food for rumen micro- .flora. An improved use of compounds derived from urea is described in the copending application of -M. F. Gribbins, S. N. 196,707, filed November 20, 1950. In theuse of urea as a source of usable nitrogen, care must be exercised to control carefully the amount of urea available to the It has been found that urea and its derivatives can be used to supply A; to and preferably about of the protein requirements.

Inasmuch as, however, large quantities of urea per se under certain conditions are broken down by microorganisms and amidases present in the rumen of certain animals, giving free ammonia, care must be exercised to prevent excess formation of the ammonia in toxic concentrations. (J. S. Dinning and H. M. Briggs Am. J. Physiol. 153, 41-46, 1948.)

An object of the present invention is to provide a new source of usable nitrogen to supplement the protein feed for the microflora of Ruminantia. Yet another object is to provide from the nontoxic derivatives of the amidines, as sources of nitrogen, a supplemental feed in which the usable synthetic nitrogen ration of the animal may be increased and the potential hazards resulting from ammonia evolution minimized. Still another object is to provide a supplemental feed composition for cattle containing, as an essential ingredient thereof, certain organic and inorganic saltsof the amidines. A further object is to provide amidine derivatives readily available for utilization by rumen microorganisms. Otherobjects and advantages of the invention will hereinafter appear.

In accord with the invention it has been found that non-toxic amidines and their derivatives can be used as a source of nitrogen to supplement protein feed of Ruminantia with a minimum requirement for restricting the ingestion of the amidines by the animal. While the said amidines break down more slowly to provide nutrients for the rumen microorganisms in their digestion of roughages, and more particularly cellulose in its various forms, the breakdown rate of these amidines to ammonia is considerably less than that from other synthetic nitrogen compounds such as urea, for example.

Work with cattle and sheep, in order to determine the value of feeds generally, has been conducted until recently directly by feeding the animal. Within the past few years an in vitro rumen and technique for its use have been developed whereby the changes taking place in feeds, brought about by microorganisms, can be measured more readily and accurately. This technique involves the use of an apparatus in which the digestive processes of the animal are conducted and can be studied in vitro. By this means, various animal feeds are introduced into or withdrawn from the laboratory unit under carefully controlled conditions and the changes taking place studied critically and progressively during the consumption of the feed by the microorganisms.

The results obtained by use of the aforesaid transplanted rumen technique have been confirmed by actual feeding of animals. This technique has established, for example, that protein addition to the feed substantially increases the consumption of cellulose by the animals and that also an increase in usable nitrogen to supplement protein feeds likewise improves ability to consume cellulose. The aforesaid and other results were determined first by laboratory experiments-working in vitro and were later established in vivo by direct feeding of animals.

The feed supplements of this invention are the amidines which not only supply a source of biologically available nitrogen as a nutrient for rumen microflora but also give a limited evolution of ammonia during ingestion. The low alfalfa :hay and salt mixture.

mixture being diluted to 900 cc. with distilled Water.

The nutrient salt solution contained a mixture of metal salts or the following composition;

Grams sodium phosphate, "mono asimsl-auvru 52.50 s dium 'mcar nat ,-s .50 37:50

v Magnesium sulfate l 2.25 Qalcium chloride 0.75 Ferrous sulfate 0.15

Rumen inoculum was obtained from the first stomach ofxa fistulated steer which had been fed with '.;a diet consisting of :grain concentrate, The extracted mumen-content was filteredithrough four thickmesses 0f cheesecloth or muslin of-comparable porosity :to remove solid matter, the liquid filtrate being employed as the ,rumen inoculum. During the removal andfiltration of the :rumen contents care was exercised to exclude air.

'ThefErlenmeyer flask wascharged with 450 cc. of this rumen liquid :to which was added 450 cc. of a distilled water suspension or solution of the aforementioned basal medium plus the nutrient 'to be evaluated. This flask was placed in a constant temperature water bath maintained at approximately 39 C. and carbon dioxide pumped through .the charge in the flask to maintain anaerobic conditions.

At .the end of the first 24 hours of bacterial digestion a sample was taken for analysis -to determine ammoniacellulose digestion and bacterial growth. After '24 hours digestion new nutrients in water were inoculated with half (e50 10,0.) of the previous day's fermentation and diluted to 900' ml. with water. The analyses and isubculturing into new nutrients was repeated over-a 4 day period.

In'the examples which follow comparisons are made between certain amidines and urea. These comparisons show the'cellulose digestion as well as the ammonia evolution of the compositions examined compared with a blank composition containing only cellulose, molasses ash, nutrient .salt solutionancl rumen inoculum.

Example 1 FLAs CONTENTS Cellulose, g

Molasses ash, g. Nutrient salt soln ml Dextrose, g.

450 450 450 dilute to 900 I111.

HEIGHT OF NH: (Mg.)

6 Atend of 19 148 19 11 135 17 0 174 20 4 1 162 51 s 155 27 ,GELLULOSE ZDIGES'IION (PERCENT) l5 atgentl oflA-Y'a-mu-v,wspam-"mm-vlfi .28, .6, 1

EmampZerZ .FLASK CONTENTS Cellulose, g Molasses ash, gr Nutrient salt soln ljgextrosag. rea,g Creotiu'nc,g A-

,Rumen moculum, ml

20 in-n.nvv .,.-..,..,.w., l-,-

"'HjEI GET '01 NH? (M2) Atcn' oi. 7

1 ys"saw-Mnqwflnsssm-mn 79.. 2.70 .11 2nd day. 10 244 47 3rd day 5: 17B 154 Ath day a 127, 125

OELLULOSE DIGESTIONKPERCENI") .Ewample 3 FLASK CONTENTS Cellulose, g 9 9 9 0 Molasses ash, g. 0.6 0.6 0.6 Nutrient salt-soln ml 70 70 l 1 1 a e 0.4 Guauidme carbonate, g. 0. 4 Rumen inoculum,-ml... 450 450 450 H2O dilute to 9001111.

HEIGHT OF 'NHa (Mg) OELLULOSE DIGESTION (PERCENT) Analysis of the digested product of Example 1 reveals that the ammonia evolution decreased from the first to the end of the 4th day from 19 to 1 mg. while the cellulose digestion averaged 28% for the 4 days, the greatest digestion occurring on the first day, tapering off to substantially nothing at the end of the 4th day. With urea as the nitrogen supplement the ammonia evolution in Example 1 averaged 155 mg. and the cellulose digestion 66%. While the cellulose digestion is augmented by the presence of urea, it is significant that the ammonia evolution is quite high. These factors are in contrast with a rumen inoculum containing creatine in which the ammonia evolution averaged but 27 mg. while the cellulose digestion averaged substantially as much as that obtained with urea as the nitrogen supplement. Similarly in Example 2 it will be noted that the ammonia evolution for creatinine averages only 99 mg. while the cellulose digestion averaged 49%. In Example 3 guanidine carbonate is compared with urea and with a rumen inoculum containing no nitrogen supplement. This amidine is superior to urea with regard to ammonia evolution, an average of only 14 mg. being evolved for the 4 day period while cellulose digestion averaged 67%, the same as that obtained with urea.

The amidines that may be used in accord with the invention as nitrogen supplements for feeding rumen microfiora, and through them aid in cellulose digestion by Ruminantia, include the non-toxic derivatives of the amidines or compounds having the amidine group C(:NH) NI-Iz and especially those compounds having the RtR group in which the R groups are hydrogen, alkyl, aryl, or ester groups. Salts of the amidines are particularly effective nitrogen supplementary feeds for rumen microfiora, such as the carbonic, acetic, hydrochloric, sulfuric and phosphoric salts.

In the formulation of feeds for cattle and. sheep, the amidine derivatives can be used to supply from 5 to 90% of the nitrogen content of the protein requirements for the animal. This is a general rule that can be followed for complete rations for the production of protein concentrate mixtures. or for the preparation of pelleted products. In the preparation of such formulated feeds, it is desirable that the synthetic nitrogen constituents thereof be preferably dry mixed with the other feed ingredients so that they can be evenly distributed throughout the feed. Any suitable mixture may be employed as, for example, a 14% protein feed can be made into an 18% feed composition by the addition of slightly more than 27 pounds of guanidine carbonate per ton of feed. The usual type of feed composition is employed to provide the normal amount of carbohydrate content which may be supplied by wheat, oats, barley, corn, hominy,

6 molasses and the like. These feeds may also contain oil meals and similar meals to supply natural proteins. For example, a typical 20% dairy ration for cattle may contain 300 lbs. of wheat bran, 310 lbs. of hominy feed, 200 lbs. of crimped oats, 300 lbs. of distillers dried grains, 175 lbs. of corn gluten feed, 400 lbs. of cocoanut oil meal, 10 lbs. of soybean oil meal, 150 lbs. of mo1asses, 200 lbs. of guanidine carbonate as an example of an amidine or derivative, 17 lbs. of brewers dried grains and 50 lbs. of minerals and salt.

Cattle are usually supplied with a diet of grass, hay and the like, augmented by a food concentrate such as that described in the last paragraph. These feed concentrates may contain from 10 to 50% protein, a small amount of minerals and the remainder mainly carbohydrate. The minerals may be added directly to the feed concentrate. When preparing the concentrate in this manner such minerals, as e. g., defiuorinated phosphate rock, salt and the like may be used. Contrariwise, the minerals may be indirectly added with the carbohydrate and/or protein as a normal part of those ingredients. In preparing such feed concentrates it has been found that the amidines hereinbefore described may constitute from 5 to of the nitrogen content of the feed, the remainder of the nitrogen being provided by protein.

The invention is not limited to the specific compositions described, nor to their specific proportions for the rate of utilization and its efliciency, as will be appreciated by those skilled in the art, is determined in large measure by the availability of the nitrogen to the rumen microorganisms and as a consequence, the percentage ingestion of a given amidine or amidine derivative that can be used varies over a wide range in accordance with the nitrogen content of that feed supplement.

I claim:

1. A supplemental feed for rumen microfiora which comprises, as a supplementary source of nitrogen other than protein, an amidine of the group consisting of creatine, creatinine, and the salts of guanidine.

2. A ruminant feed composition suitable for the in vivo and in vitro feeding of rumen microfiora containing a non-toxic derivative of an amidine containing biologically available nitrogen.

3. A ruminant feed composition suitable for the in vivo and in vitro feeding of rumen microflora containing carbohydrates, protein and as a supplementary source of nitrogen a non-toxic derivative of an amidine.

4. A ruminant feed composition containing carbohydrates, minerals, protein and as a supplementary source of nitrogen a non-toxic derivative of an amidine containing biologically available nitrogen which supplies from 5 to 90% of the nitrogen content of the feed.

5. A ruminant feed composition containing from 10 to 50% protein, minerals and the remainder mainly carbohydrate products, a nontoxic derivative of an amidine containing biologically available nitrogen supplying from 5 to 90% of the nitrogen content of the feed.

6. A ruminant feed composition suitable for the in vivo and in vitro feeding of rumen microfiora containing carbohydrates, protein and creatine as a supplementary source of nitrogen.

7. A ruminant feed composition suitable for the in vivo and in vitro feeding of rumen microfiora containing carbohydrates, protein and creatinine as a supplementary source of nitrogen.

in vivo and in vitro feeding of rumen microflora containing carbohydrates, protein and an amidine salt as a supplementary source of nitrogen.

10. A ruminant feed composition suitable for the in vivo and in vitro feeding of rumen micro- 1 flora containing carbohydrates, protein and an am dine alt of an acid of the group carbonic,

acetic, hydrochloric, sulfuric and phosphoric acids as a supplementary source of nitrogen.

KENNETH E. WALKER.

5 REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS 0 Number Name Date 2,522,854 Brink et a1 Sept. 19, 1950 2,560,830 Turner July 17, 1951 

1. A SUPPLEMENTAL FEED FOR RUMEN MICROFLORA WHICH COMPRISES, AS A SUPPLEMENTARY SOURCE OF NITROGEN OTHER THAN PROTEIN, AN AMIDINE OF THE GROUP CONSISTING OF CREATINE, CREATININE, AND THE SALTS OF GUANIDINE. 